Preparation of pancreatic cancer dendritic cell vaccine and application of pancreatic cancer dendritic cell vaccine in treatment of pancreatic cancer

Abstract

The invention relates to preparation of a pancreatic cancer dendritic cell vaccine and application of the pancreatic cancer dendritic cell vaccine in treatment of pancreatic cancer, relates to the technical field of biological medicines, and comprises a preparation method of Panc-1 and AsPC-1 lysates, a preparation method of a DC vaccine loaded with the Panc-1 and AsPC-1 lysates and function verification of DC cells. The DC vaccine prepared by the invention not only stimulates T cells to generate more IFN-gamma, but also induces to generate stronger tumor killing T cells, and provides a new thought for research and development of pancreatic cancer DC vaccines. In addition, Panc-1 and AsPC-1 lysates are adopted as antigens of the DC vaccine, so that the problem that autologous tumor antigens are difficult to obtain is solved. Furthermore, Panc-1 and AsPC-1 lysates are adopted as antigens of the DC vaccine, and the mixed lysates contain abundant and wide tumor antigens and can prevent immune escape of tumors.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the preparation of a pancreatic cancer dendritic cell vaccine and its application in the treatment of pancreatic cancer. Background Art

[0002] Pancreatic cancer is a highly malignant tumor with a five-year survival rate of less than 7%. It is predicted that by 2030, the mortality rate of pancreatic cancer will rise to the second place. Most patients have local infiltration and distant metastasis at the time of first diagnosis, which has become the main factor for the high mortality rate of pancreatic cancer. Currently, surgical resection is the most effective treatment for pancreatic cancer, but due to the high invasiveness and high metastasis of the tumor, even after surgery, more than 76% of recurrent patients will have distant metastasis of the tumor. In addition, pancreatic cancer is relatively insensitive to chemotherapy and radiotherapy.

[0003] In recent years, immunotherapy has been proven to be another important anti-tumor method in addition to surgery and chemotherapy. Immunotherapy is a treatment method that fights tumors by activating the human immune system. It can kill tumor cells and control the development of tumors by stimulating and enhancing the patient's immunity. At present, immunotherapy for pancreatic cancer mainly includes treatment methods such as immune checkpoint inhibitors, adoptive T cell therapy, oncolytic viruses, and specific immunomodulators. However, clinical experimental results show that the above immunotherapy methods have failed to significantly prolong the patient's overall survival and progression-free survival. In comparison, DC vaccines have shown relatively good therapeutic effects in clinical studies on the treatment of pancreatic cancer and can prolong the patient's overall survival. As the most important antigen-presenting cell in the body, DC vaccines can process and present tumor antigens to the surface of T cells, thereby producing tumor-specific cytotoxic T lymphocytes.

[0004] In the prior art, there have been some reports on the use of DC vaccines to treat pancreatic cancer. DC vaccines are designed to force the presentation of tumor antigens to the patient's immune system. This strategy usually involves the use of DCs prepared from patient-derived monocytes, loaded with determined tumor antigens or protein lysates, activated in vitro with cytokines and adjuvants to prepare DC vaccines, which are then re-infused into the patient's body to achieve anti-tumor and anti-viral purposes. Relevant targets for the treatment of pancreatic cancer include CLDN18, WT1, MUC1, CEACAM5, MSLN, etc. In order to gain a deeper understanding of the expression differences of tumor-associated antigens in pancreatic cancer, we compared the gene transcription levels of CLDN18, WT1, MUC1, CEACAM5, and MSLN in pancreatic cancer tissues and adjacent tissues. Figure 1 As shown, the expression of five genes in pancreatic cancer tissues was significantly higher than that in adjacent tissues, and the results of database bioinformatics analysis were consistent with literature reports.

[0005] The antigens loaded by DC mainly include tumor-associated antigen peptides and pancreatic cancer whole cell lysates. The antigen peptides are mainly WT1 and MUC1. The pancreatic cancer cell lysates mainly include pancreatic cancer cell lysates, patient pancreatic cancer stem cell lysates, and tumor cells expressing α-Gal epitope. Studies have shown that the use of WT1 or WT1\MUC1 loaded DC cells can significantly prolong the overall survival time and progression-free survival of pancreatic cancer patients after surgery. Hanada et al. used DC cells loaded with WT1 to treat tumor patients after pancreatic cancer resection. A total of 6 patients had a progression-free survival of 19.9 months and an overall survival of 59 months. Nagai, K et al. used DC cells loaded with WT1\MUC1 to treat tumor patients after pancreatic cancer resection. A total of 10 patients had a progression-free survival of 17.7 months and an overall survival of 46.4 months. However, the two peptides alone are relatively simple and it is difficult to cover most of the tumor antigens of pancreatic cancer. Immune escape is one of the key mechanisms for inducing tumors, and antigen peptides as vaccine antigens are limited by the patient's MHC type, which means that not all patients can benefit from them.

[0006] Furthermore, we queried the CCLE database for the gene expression of tumor-associated antigens such as CLDN18, WT1, MUC1, CEACAM5, and MSLN in the tumor cell lines Panc-1 and AsPC-1 that were established by in vitro culture of tumor cells from pancreatic cancer patients and could be stably propagated. The information in the database showed that both cell lines had high transcriptional expression levels, such as Figure 2 We used a combination of cell lines as DC loads to generate a higher immune response.

[0007] In addition to in vitro experiments, pancreatic cancer cells also contain a large number of tumor-associated antigens and specific antigens. At present, some studies have tried to use modified whole pancreatic cancer cells as vaccine antigens, such as GVAX, which is a pancreatic cancer cell that has been irradiated with gamma rays and transduced with the GM-CSF gene. GVAX can secrete GM-CSF, attract and activate dendritic cells (DCs), and thus induce antigen-specific T cell responses. Although the vaccine has performed well in terms of safety and tolerability, it has not yet shown superior efficacy compared to existing standard treatments.

[0008] Furthermore, allogeneic tumor cell lysates were used to load autologous DCs, which were able to induce tumor-specific T cell responses in pancreatic cancer patients. +Using pancreatic cancer cell Panc-1 lysate to load DC can generate a specific T cell immune response against pancreatic cancer. However, due to the heterogeneity of pancreatic cancer, a single Panc-1 lysate is difficult to cover more tumor antigens, and needs to be used in combination with other tumor lysates or tumor antigen peptides to produce a higher immune response. Summary of the invention

[0009] In view of the shortcomings of the prior art, the purpose of the present invention is to propose a preparation of a pancreatic cancer dendritic cell vaccine and its application in the treatment of pancreatic cancer. The specific scheme is as follows:

[0010] A preparation method of a pancreatic cancer dendritic cell vaccine comprises the following steps:

[0011] 1) Take equal amounts of Panc-1 and AsPC-1 cells and place them in cryopreservation tubes, and freeze and thaw the cells repeatedly at low and normal temperatures;

[0012] 2) The cells were ultrasonically disrupted and centrifuged respectively, the supernatant was collected, and sterilized and filtered. The filtrate was the cell lysate. The protein concentration was detected by BCA method and the protein concentration was adjusted;

[0013] 3) CD14 + The cell suspension was transferred to a six-well plate, with 2x10 5 -2x10 6 cells, a volume of 1 mL, and recombinant GM-CSF and recombinant IL-4 were added to a six-well plate;

[0014] 4) Place the six-well plate on a clean bench and gently shake it three times to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days.

[0015] 5) Add recombinant GM-CSF (final concentration of 400-4000 IU / mL) and recombinant IL-4 (final concentration of 200-1000 IU / mL) to the six-well plate;

[0016] 6) The cells were placed in an incubator at 37°C and 5% CO2 for 2 days to obtain immature dendritic cells; appropriate amounts of Panc-1 and AsPC-1 (final concentrations were 50 μg / mL to 3000 μg / mL) were added and mixed with the immature dendritic cells for 6 hours;

[0017] 7) Then add 2 μL of TNF-α (final concentration of 400-4000 IU / mL), 2 μL of LPS (final concentration of 1-5 μg / mL) and 1 μL of Poly (I:C) (final concentration of 0.5-5 μg / mL) to stimulate dendritic cells for 24 hours to induce DC cell maturation. The mature cells are the DC vaccine.

[0018] The DC vaccine prepared by the above technical solution not only stimulates T cells to produce more IFN-γ, but also induces the production of stronger tumor-killing T cells, providing a new idea for the development of pancreatic cancer DC vaccine. In addition, the use of Panc-1 and AsPC-1 lysates as DC vaccine antigens solves the problem of the difficulty in obtaining autologous tumor antigens. Furthermore, Panc-1 and AsPC-1 lysates are used as DC vaccine antigens. The mixed lysate contains a rich and extensive tumor antigen, which can prevent tumor immune escape.

[0019] Furthermore, the low temperature in step 1) is -60°C to -200°C.

[0020] Furthermore, the normal temperature in step 1) is 34°C to 38°C.

[0021] Furthermore, the number of freeze-thaw cycles in step 1) is 3 to 6 times.

[0022] Furthermore, the ultrasonic crushing time in step 2) is 1 to 10 minutes.

[0023] Furthermore, the centrifugal speed in step 2) is 1000-3000 rpm.

[0024] Furthermore, the centrifugation time in step 2) is 5 to 20 minutes.

[0025] Furthermore, in step 6), appropriate amounts of Panc-1 and AsPC-1 cell lysates prepared in step 1 are added respectively. The lysates may be Panc-1 or Panc-1 and AsPC-1 cell lysates.

[0026] Furthermore, the final concentration of the Panc-1 and AsPC-1 cell lysis mixture proteins added in step 6) is 50 μg / mL to 3000 μg / mL.

[0027] Use of a pancreatic cancer dendritic cell vaccine as described in any one of the above items in the treatment of pancreatic cancer.

[0028] In summary, the present application includes the following at least one preparation of a pancreatic cancer dendritic cell vaccine and its application in the treatment of pancreatic cancer and beneficial technical effects:

[0029] In use, the prepared DC vaccine not only stimulates T cells to produce more IFN-γ, but also induces the production of stronger tumor-killing T cells, providing a new idea for the development of pancreatic cancer DC vaccines. In addition, the use of Panc-1 and AsPC-1 lysates as DC vaccine antigens solves the problem of the difficulty in obtaining autologous tumor antigens. Furthermore, the use of Panc-1 and AsPC-1 lysates as DC vaccine antigens, the mixed lysate contains a rich and extensive tumor antigen, which can prevent tumor immune escape. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a comparison of the expression of five genes in pancreatic cancer and adjacent tissues;

[0031] Figure 2 is the expression level of the five genes in Panc-1 and AsPC-1 cell lines;

[0032] Figure 3 This application mainly provides CD3 + The level of INFγ produced by T cells;

[0033] Figure 4 This application mainly provides CD3 + Cytotoxic activity of T cells against pancreatic cancer cells (Panc-1). DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, objectives and effects of the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments.

[0035] The following is combined with Figure 3-4 This application is described in further detail.

[0036] The present application discloses the preparation of a pancreatic cancer dendritic cell vaccine and its application in the treatment of pancreatic cancer.

[0037] Example 1

[0038] A preparation method of a pancreatic cancer dendritic cell vaccine comprises the following steps:

[0039] 1) Take equal amounts of Panc-1 and AsPC-1 cells respectively and put them into cryopreservation tubes, and freeze and thaw the cells repeatedly under low temperature and room temperature, the low temperature is -60°C to -200°C, preferably, the low temperature is -180°C. The room temperature is 34°C to 38°C, preferably, the room temperature is 37°C. The number of multiple freeze-thaw cycles is 3 to 6 times, preferably, the number of multiple freeze-thaw cycles is 5 times.

[0040] 2) Ultrasonic disruption and centrifugation of cells respectively, collecting supernatant, sterilizing and filtering, the filtrate is cell lysate, detecting protein concentration by BCA method, and adjusting protein concentration. Ultrasonic disruption time is 1 to 10 minutes, preferably, ultrasonic disruption time is 5 minutes. Centrifugal speed is 1000 to 3000 rpm, preferably, centrifugal speed is 1500 rpm. Centrifugal time is 5 to 20 minutes, preferably, centrifugal time is 10 minutes.

[0041] 3) CD14 + The cell suspension was transferred to a six-well plate, with 2x10 5 -2x10 6 cells in a volume of 1 mL, and recombinant GM-CSF and recombinant IL-4 were added to the six-well plate. GM-CSF is a granulocyte-macrophage colony-stimulating factor, which is a cytokine secreted by a variety of cells (such as T cells, macrophages, endothelial cells, etc.) and plays an important regulatory role in the production and function of blood cells. IL-4 is an important cytokine that belongs to the interleukin family. It is secreted by activated T helper cells (mainly Th2 cells), mast cells, basophils, and certain types of dendritic cells.

[0042] The final concentration of recombinant GM-CSF is 400-4000 IU / mL, preferably, the final concentration of recombinant GM-CSF is 2000 IU / mL. The final concentration of recombinant IL-4 is 200-1000 IU / mL, preferably, the final concentration of recombinant IL-4 is 800 IU / mL.

[0043] 4) Place the six-well plate on a clean bench and gently shake it three times to evenly disperse the cells. Incubate at 37°C and 5% CO2 for 3 days.

[0044] 5) Add recombinant GM-CSF and recombinant IL-4 to the six-well plate.

[0045] 6) The cells were placed in an incubator at 37° C. and 5% CO 2 for 2 days to obtain immature dendritic cells; appropriate amounts of Panc-1 and AsPC-1 cell lysates were added and mixed, and co-cultured with the immature dendritic cells for 6 hours.

[0046] Add an appropriate amount of prepared Panc-1 or AsPC-1 cell lysate, which may be Panc-1 or Panc-1 or AsPC-1 cell lysate. Preferably, the lysate may be Panc-1 or AsPC-1 cell lysate. The final concentration of the Panc-1 or AsPC-1 cell lysate mixture protein is 50 to 3000 μg / mL, and preferably, the final concentration of the Panc-1 or AsPC-1 cell lysate mixture protein is 1500 μg / mL.

[0047] 7) Then, TNF-α (final concentration of 400-4000 IU / mL), LPS (final concentration of 1-5 μg / mL) and Poly(I:C) (final concentration of 0.5-5 μg / mL) were added to stimulate dendritic cells for 24 hours to induce DC cell maturation. The mature cells are the DC vaccine.

[0048] Preferably, the dosage of TNF-α is 2 μL, and the final concentration is 2000 IU / mL, the dosage of LPS is 1 μL, and the final concentration is 3 μg / mL, and the dosage of Poly(I:C) is 1 μL, and the final concentration is 3 μg / mL.

[0049] LPS is a complex molecule present in the cell wall of Gram-negative bacteria and is an important endotoxin. LPS is of great significance in immunology and microbiology, mainly because it can strongly activate the immune system and induce inflammatory responses. Preferably, the final concentration of LPS is 2 μg / mL.

[0050] Poly(I:C) is a long double-stranded RNA molecule composed of hypoxanthine and cytidine. This structure mimics the double-stranded RNA of certain viruses, so it can effectively activate the immune system and is used to study immune responses. Preferably, the final concentration of Poly(I:C) is 1 μg / mL.

[0051] Isolation of peripheral blood mononuclear cells:

[0052] Step 1: Add 4.5 mL of Paque Plus solution;

[0053] Step 2: Use a pipette to draw blood from a pancreatic cancer patient and slowly inject it along the wall of the centrifuge tube. Paque Plus solution upper layer, 10 mL per tube. Centrifuge at 800 g for 20 min at room temperature; Paque Plus solution is a density gradient medium commonly used for cell separation. It is commonly used in laboratory techniques such as cell separation and blood sample processing, especially in immunology and cell biology research.

[0054] Step 3: Take out the centrifuge tube, and the sample is divided into four layers, from top to bottom: plasma, monocytes, Ficoll solution, red blood cells and granulocytes;

[0055] Step 4: Carefully aspirate the mononuclear cell layer and transfer it to a 15mL centrifuge tube, add PBS / 1% FBS solution to make up to 14mL, and mix by pipetting. Centrifuge at room temperature at 800g for 5 minutes; In the prior art, PBS / 1% FBS solution is a commonly used cell treatment and experimental buffer, which is designed to maintain the physiological state and activity of cells and reduce possible cell damage during the experiment.

[0056] Step 5: Discard the supernatant, flick the bottom of the tube to loosen the cells, add 14 mL of PBS / 1% FBS solution to resuspend the cells. Centrifuge at 700 g for 5 min at room temperature;

[0057] Step 6: Discard the supernatant and flick the bottom of the tube to loosen the cells. Add 14 mL of RPMI / 10% FBS solution to resuspend the cells. Centrifuge at 400 g for 5 min at room temperature; In the prior art, RPMI / 10% FBS solution is used to resuspend cells. Here, "RPMI" refers to RPMI 1640 culture medium, and "10% FBS" refers to the culture medium with 10% fetal bovine serum added.

[0058] Step 7: Remove the supernatant and flick the bottom of the tube to loosen the cells. Add 10 mL of RPMI / 10% FBS solution to resuspend the cells, pipette to mix, and count the cells;

[0059] Step 8: Centrifuge at room temperature, 700 g for 5 min, discard the supernatant, and add appropriate amount of PBS / 1% FBS for subsequent experiments. + Monocytes: This example uses EasySep TM CD14 positive selection kit.

[0060] Step 9: Transfer the mononuclear cell population separated from the peripheral blood to a 5 mL flow tube;

[0061] Step 10: Add an appropriate amount of selection cocktail solution to a final concentration of 100 μL / mL, pipette and mix thoroughly, and incubate at room temperature for 10 minutes; in the prior art, selection cocktail solution is a solution used to select or enrich a specific cell population.

[0062] Step 11: Prepare CD14 positive selection beads and place RapidSphere TM The solution was vortexed for 30 seconds to evenly disperse the magnetic beads. TM A solution is a commercial reagent or solution used for cell separation and enrichment. It is a density gradient separation medium designed to provide a fast and efficient cell separation solution.

[0063] Step 12: Add appropriate amount of RapidSphere to the flow tubeTM The solution was adjusted to a final concentration of 100 μL / mL, mixed thoroughly by pipetting, and incubated at room temperature for 3 min.

[0064] Step 13: Add an appropriate amount of PBS / 2% FBS with 1mM EDTA solution to the flow tube to a total volume of 2.5 mL, and mix thoroughly by pipetting; in the prior art, PBS / 2% FBS with 1mM EDTA solution is a buffer solution commonly used in cell biology research, mainly used for cell processing and separation.

[0065] Step 14: Insert the flow tube vertically into the EasySep TM magnet, incubate at room temperature for 3 minutes; in the prior art, EasySep TM A magnet is a device used for cell separation, designed to isolate and enrich specific types of cells efficiently and easily.

[0066] Step 15: Invert the magnet and collect the cell fluid flowing out of the flow tube into a 15 mL centrifuge tube. Keep the magnet inverted for 3 seconds. Do not shake or absorb the liquid on the tube wall.

[0067] Step 16: Place the magnet upright and remove the flow tube.

[0068] Step 17: Repeat Step 15 and Step 16 twice.

[0069] Step 18: Add 2 mL RPMI / 10% FBS to the flow tube to resuspend the cells, stain with Trypan Blue, and count the cells.

[0070] Induction of immature dendritic cells:

[0071] Step 1: Place CD14 + The cell suspension was transferred to a six-well plate, with 2×10 5 -2×10 6 cells, the volume is 1 mL, recombinant GM-CSF and recombinant IL-4 are added to the six-well plate, the final concentration of recombinant GM-CSF is 400-4000 IU / mL, preferably, the final concentration of recombinant GM-CSF is 2000 IU / mL. The final concentration of recombinant IL-4 is 200-1000 IU / mL, preferably, the final concentration of recombinant IL-4 is 800 IU / mL.

[0072] Step 2: Place the six-well plate on a clean bench and gently shake it three times to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days.

[0073] Step 3: Add recombinant GM-CSF and recombinant IL-4 to the six-well plate, the final concentration of recombinant GM-CSF is 400-4000 IU / mL, preferably, the final concentration of recombinant GM-CSF is 2000 IU / mL. The final concentration of recombinant IL-4 is 200-1000 IU / mL, preferably, the final concentration of recombinant IL-4 is 800 IU / mL.

[0074] Step 4: Place the cells in an incubator at 37° C. and 5% CO 2 for 2 days to obtain immature dendritic cells.

[0075] Preparation of cell lysate:

[0076] Step 1: Resuspend Panc-1 and AsPC-1 in RPMI1640 at a density of 5×10 6 / mL, the volume is 1mL.

[0077] Step 2: Freeze the cells in liquid nitrogen for 20 seconds and immediately thaw the cells completely in a 37°C water bath.

[0078] Step 3: Repeat step 2 5 times.

[0079] Step 4: Centrifuge the repeatedly frozen and thawed cells, collect the supernatant, detect the protein concentration, and prepare to load DC cells.

[0080] Induction of mature dendritic cells:

[0081] Immature DC cells were divided into 4 groups, namely, the group without antigen loading, the group loaded with Panc-1 lysate, the group loaded with AsPC-1 lysate, and the group loaded with Panc-1 and AsPC-1 lysate mixture.

[0082] Different groups of antigens are added to the corresponding immature dendritic cells, and the final concentration of the Panc-1 lysate histone loaded alone is 20-200 μg / mL. Preferably, the final concentration of the Panc-1 lysate histone loaded alone is 100 μg / mL.

[0083] The final concentration of AsPC-1 lysate histones loaded alone is 20-200 μg / mL, preferably, the final concentration of AsPC-1 lysate histones loaded alone is 120 μg / mL. The final concentration of the mixed protein of Panc-1 and AsPC-1 lysate is 20-200 μg / mL, preferably, the final concentration of the mixed protein of Panc-1 and AsPC-1 lysate is 90 μg / mL.

[0084] After 6 hours of culture, TNF-α (final concentration of 400-4000 IU / mL) and LPS (final concentration of 1-5 μg / mL) were added to stimulate the dendritic cells for 24 hours to induce DC cell maturation, and the mature cells were DC vaccines. Preferably, the final concentration of TNF-α is 2000 IU / mL, the final concentration of LPS is 3 μg / mL, and the final concentration of Poly(I:C) is 2 μg / mL.

[0085] Example 2

[0086] Functional verification of DC cells

[0087] T cell preparation:

[0088] Step 1: Count PBMC cells, centrifuge at 300g for 5 min, remove the supernatant, add appropriate amount of PBS / 1% FBS to resuspend the cells to 1×10 8 / mL, transfer to a 5mL sterile flow tube. The volume added to the flow tube at one time should be between 0.1-2mL.

[0089] Step 2: According to the instructions of the magnetic separation kit, add a suitable volume of Selection Cocktail, mix thoroughly, and incubate at room temperature for 5 minutes. In the prior art, Selection Cocktail can promote cell growth.

[0090] Step 3: Dextran RapidSpheres TM Vortex for 30 seconds and add the corresponding volume of Dextran RapidSpheres according to the instructions of the sorting kit. TM , mix thoroughly, and incubate at room temperature for 3 minutes. TM It is a magnetic bead product used in magnetic separation technology. These magnetic beads are usually coated with dextran and have a magnetic core. This design allows them to be used in a variety of biological separation and purification applications.

[0091] Step 4: Add PBS / 2% FBS with 1mM EDTA solution to the flow tube to a total volume of 2.5mL. Mix thoroughly and insert the flow tube vertically into the EasySep TM Magnet, incubate at room temperature for 3 minutes. EasySep TM Magnet is a magnetic rack device used for cell sorting. It uses magnetic separation technology to separate specific types of cells from mixed cell populations.

[0092] Step 5: Invert the magnet and collect the cell fluid flowing out of the flow tube into a 15mL centrifuge tube. Keep the magnet inverted for 3 seconds without shaking or absorbing the liquid on the tube wall; turn the magnet upright and take out the flow tube.

[0093] Step 6: Repeat steps 4 to 5 twice

[0094] Step 7: Add 2 mL of RPMI / 10% FBS to the flow tube to resuspend the cells for counting and subsequent experimental operations.

[0095] DC activation in vitro CD3 + T cells:

[0096] The experiment was divided into four groups, namely negative control group (DC without antigen loading), experimental group 1 (DC loaded with Panc-1 lysate), experimental group 2 (DC loaded with Panc-1 lysate), and experimental group 3 (DC loaded with Panc-1 and AsPC-1 lysate), with 3 replicates in each group. The DC density was adjusted to 2×10 5 / mL, adjust autologous CD3 + The density of T lymphocytes was 2 × 10 6 / mL.

[0097] First put CD3 + T cells were inoculated in a 96-well plate, and DCs were added to CD3 T cells at a ratio of 1:10 (DC:T) on D0, D7, D14, and D21. + T cells, IL-2 (10-100 units / mL) is added at the same time. It is the abbreviation of interleukin-2. It is a cytokine secreted by T cells and plays an important regulatory role in the immune system. Preferably, the activity unit of IL-2 per milliliter is 80 units / mL. IL-2 and IL-7 (1-10 ng / mL), preferably, IL-7 is 6 ng / mL. After the second stimulation for 48 hours, the cell culture supernatant was collected and the level of INFγ in the supernatant was detected by ELISA. After DC stimulation for 4 times, CD3 + T cells were used to detect their cytotoxic activity against pancreatic cancer cells (Panc-1).

[0098] Reference Figure 1 ELISA method to detect CD3 + T produces the level of INFγ:

[0099] Add the cell culture supernatant from the above to the reaction wells (100 μl / well), seal the reaction wells with sealing tape, incubate at 37°C for 90 minutes, and wash the plate 4 times. Add 100 μl of horseradish peroxidase-labeled anti-cytokine antibody. Seal the plate and incubate at room temperature for 60 minutes. Wash the plate 4 times and buckle dry. Add enzyme conjugate working solution (100 μl / well), seal the reaction wells with sealing tape, incubate at 37°C for 30 minutes, and wash the plate 4 times. Add 100 μl / well of color development solution and incubate at 37°C for 15 minutes in the dark. Add 100 μl / well of stop solution, mix well and measure OD immediately 450 The concentration of cytokines was calculated according to the standard curve formula.

[0100] Reference Figure 2 , CD3 + Cytotoxic activity of T cells against pancreatic cancer cells (Panc-1):

[0101] The CD3 + T cells were added to a 96-well plate, with 8 × 10 cells per well. 5 , 4×10 5 , 1×10 5 1×10 4 CSFE-labeled Panc-1, CD3 + The ratio of T cells to Panc-1 was 80:1, 40:1, and 10:1, respectively. CSFE is a fluorescent dye used to label the cytoplasm of living cells. After 10 hours of culture, cells in each well were collected and stained with Annexin V. Annexin V is a protein that is widely used in cell biology and biomedical research, especially in the study of apoptosis (programmed cell death). It is a calcium ion-dependent phospholipid binding protein, mainly used to detect the externalization of phosphatidylserine (PS) on the cell membrane. The activity of Panc-1 cells was detected by flow cytometry, and the percentage (%) of Panc-1 specific killing was calculated.

[0102] The experimental results showed that the prepared dendritic cell vaccine can activate T cells, induce T cells to produce a large amount of INFγ, and specifically kill pancreatic cancer cells (Panc-1).

[0103] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a pancreatic cancer dendritic cell vaccine, characterized in that: The steps include: 1) Take equal amounts of Panc-1 and AsPC-1 cells and place them in cryopreservation tubes, and freeze and thaw the cells repeatedly at low and normal temperatures; 2) The cells were ultrasonically disrupted and centrifuged respectively, the supernatant was collected, and sterilized and filtered. The filtrate was the cell lysate. The protein concentration was detected by BCA method and the protein concentration was adjusted; 3) CD14 + The cell suspension was transferred to a six-well plate, with 2x10 5 -2x10 6 cells, a volume of 1 mL, and recombinant GM-CSF and recombinant IL-4 were added to a six-well plate; 4) Place the six-well plate on a clean bench and gently shake it three times to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days. 5) Add recombinant GM-CSF (final concentration of 400-4000 IU / mL) and recombinant IL-4 (final concentration of 200-1000 IU / mL) to the six-well plate; 6) The cells were placed in an incubator at 37°C and 5% CO2 for 2 days to obtain immature dendritic cells; appropriate amounts of Panc-1 and AsPC-1 (final concentrations were 50 μg / mL to 3000 μg / mL) were added and mixed with the immature dendritic cells for 6 hours; 7) Then add 2 μL of TNF-α (final concentration of 400-4000 IU / mL), 2 μL of LPS (final concentration of 1-5 μg / mL) and 1 μL of Poly (I:C) (final concentration of 0.5-5 μg / mL) to stimulate dendritic cells for 24 hours to induce DC cell maturation. The mature cells are the DC vaccine.

2. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The low temperature described in step 1) is -60°C to -200°C.

3. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The normal temperature described in step 1) is 34°C to 38°C.

4. The preparation of a pancreatic cancer dendritic cell vaccine and its use in the treatment of pancreatic cancer according to claim 1, characterized in that: The number of multiple freeze-thaw cycles in step 1) is 3 to 6 times.

5. The preparation of a pancreatic cancer dendritic cell vaccine and its use in the treatment of pancreatic cancer according to claim 1, characterized in that: The ultrasonic crushing time in step 2) is 1 to 10 minutes.

6. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The centrifugal speed described in step 2) is 1000-3000 rpm.

7. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The centrifugation time in step 2) is 5 to 20 minutes.

8. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: In step 6), appropriate amounts of the Panc-1 and AsPC-1 cell lysates prepared in step 1 are added respectively. The lysates may be Panc-1 or Panc-1 and AsPC-1 cell lysates.

9. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The final concentrations of the Panc-1 and AsPC-1 cell lysis mixture proteins added in step 6) are both 50 μg / mL to 3000 μg / mL.

10. Use of a pancreatic cancer dendritic cell vaccine according to any one of claims 1 to 9 in the treatment of pancreatic cancer.

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